| Object and contents:TGF-β-activated kinase1(TAK1), a member of MAPKKK family, was first reported as a regulator of MAP kinase signaling induced by TGF-β1. TAK1mRNA was present in all tissues examined, and was particularly abundant in thymus and brain, with a lesser amount in liver.TAKl can be activated by many cytokines and stress, it is a key mediator of proinflammatory and stress signals. TAKl controls downstream events linked to cell activation and programmed cell death.Meanwhile, TAK1have essential function in innate and adaptive immune response. Up till now, many papers demonstrated that TAK1is critical for the survival of many kinds of cells, included lymphocytes and neutrophils. However, the specific function, if any, of TAKl in myeloid cell development and TLR-induced innate immunity remains largely unknown.In this study,we found that LPS and TGF-β1could activated TAK1in macrophages,the overexpression of TAKl could promote the survival of macrophages.Methods and results:The project is divided into three parts:Part One:Examined the expression level of TAK1in macrophages after the stimulationwith LPS or TGF-p1.1. We detected the mRNA expression levels of TAKl in different immune cells. Results show that TAKl mRNA was present abundantly in all of these immune cells.2. We observed gene and protein expression of TAK1in macrophages after the stimulationwith LPS or TGF-β1. Results show that TAK1can be activated in a time-dependent manner.Part Two:Established the optimal methods for electroporation on RAW264.7.1. Preparation of competent cells of E. coli and transformed into the appropriate plasmid, results showed that the competent cells worked well, and we extracted high purity and concentration of plasmids; 2. We established the optimal methods for cell culture of RAW264.7. The trypsin used in conjunction with cell scraper minimized the amage of passage.For the optimal methods for transfection,the amount of plasmid is20ug,the voltage value of250V,capacitance value of960uF,resistance value of oo.3. We established RAW264.7cell lines transiently expressing target plasmids, which were confirmed by Real-time PCR and Western blotting assay of TAK1expression,and results showed that we transfected the plasmids successfully. Part Three:Preliminarily observed the influence of overexpression of TAK1on apoptosis of macrophages.We detected the apoptosis of transfected RAW264.7macrophage cells after LPS or TGF-β1treatment. Results showed that overexpression of TAK1may inhibit the effect of macrophage apoptosis induced by LPS and TGF-β1.Conclusion:Taken all data together, we demonstrate that TGF-β-activated kinase1(TAK1) can be activated by LPS and TGF-β1in macrophages.The overexpression of TAK1may promote the survival of macrophages. Our studies shed light on the role of TAK1in regulation of macrophage and LPS-activated TLR4signal transduction pathway. |
